Presently, there is no evidence for the ability of mature osteoblast lineage cells to dedifferentiate. However, if possible, de-differentiation of mature osteoblast lineage cells could provide an additional source of osteoblast population capable of proliferation and differentiation. We hypothesize that under the conditions of high demand for osteoblast lineage activity, preosteocytes and osteocytes can undergo a de-differentiation, accompanied by dramatic changes in gene expression. This process would generate cells that could exhibit proliferation potential, followed by differentiation into bone matrix producing osteoblasts. Our preliminary data support these hypotheses, and in this proposal we seek to determine whether de-differentiation of osteoblast lineage cells is an integral aspect of bone remodeling and to evaluate the changes in gene expression involved. Intermittent administration of PTH results in a bone-anabolic response, but the mechanisms involved are not completely understood. We hypothesize that PTH-induced de-differentiation of osteoblast lineage cells could contribute to this bone-anabolic response. To test these hypotheses, we propose an in vivo evaluation of dedifferentiation - redifferentiation of preosteocytes/osteocytes. Utilizing a transgenic model in which we can achieve a complete ablation of mature osteoblasts, we will evaluate the ability of de-differentiated preosteocytes/osteocytes to repopulate the osteoblast niche. In addition, the ability of de-differentiated osteocytic cells obtained from bone chip outgrowth cultures (BCOC) to re-differentiate into mature osteoblasts in vitro and in vivo, following a transplantation protocol will be assessed. We will also evaluate the effects of PTH on the osteocyte de-differentiation and changes in osteocyte gene expression utilizing an in vivo model of intermittent treatment with PTH. This will be achieved by utilizing osteocyte specific DMP-1Cre transgenic mice and ZEG (GFP reporter mouse) that would allow also for the lineage tracing of the cells following PTH treatment. Equally important will be to define the changes in expression of genes responsible for matrix removal and de-differentiation process in vivo under PTH influence. Utilizing the transgenic approach and flow cytometry we will selectively isolate the RNA from the osteocytes (DMP1 expressing cells), following an intermittent treatment with PTH. This will allow for the study of the mechanism by which the PTH exerts its effects on this mature osteoblast lineage population and induces matrix removal. PUBLIC HEALTH RELEVANCE: This proposal seeks to evaluate de-differentiation of mature osteoblast lineage cells (preosteocyte/osteocyte) and to establish this process as an integral aspect of bone remodeling. We will evaluate if treatment with parathyroid hormone could induce this de-differentiation process. This would then follow by the evaluation of the effects of various potential therapeutic agents that would be involved in regulation of de-differentiation. The knowledge developed in this grant will provide the foundation for future studies aimed at evaluating factors that would affects process of de-differentiation with the goal of increasing the bone mass in osteoporosis and other disorders with low bone mass.